Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure

Abstract

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert^® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

Keywords: Ct value; G29179T; GeneXpert; SARS-CoV-2; infection control; quality control.

Figure 1

Mutations in the nucleocapsid gene of SARS-CoV-2 from patients with COVID-19 and the strategy used to reveal the mutation responsible for the N gene (N2 target region) amplification delay in the GeneXpert^® System and Cepheid Xpert Xpress SARS-CoV-2 assay kit (GX) results. Orange bases indicate the N2 target region for Sanger sequencing of the nucleocapsid gene; the genome positions are numbered according to the reference genome (Wuhan-HU-1; NC_045512.2). The primers and probe for the US Centers for Disease Control and Prevention (CDC) primer and probe targets, the Japan National Institute of Infectious Disease (NIID) primers, and the primers designed for the present study are shown. The GX primer targets were very similar to those of the CDC-published PCR primer sets. Green and blue bases indicate the location of the 29179: G/T mutation that spans five nucleotides upstream of the 3’ end of the CDC forward primer (possibly the same as the GX primer). Yellow and blue bases indicate other mutation locations: one within the CDC reverse primer (29218: C/T) and two in regions unrelated to any primer or probe (28983: A/G; 29253: C/T). Blue-only bases indicate previously described N gene mutations on the probe binding site that resulted in GX assay failure and instability (29197: C/T; 29200: C/T or A; 29203: C/T).

Figure 2

Comparison between N2 and E Ct values analyzed through GX. A total of 354 cases with positive or presumptive positive GX results were correlated with two Ct values of N2 and E. Undetectable cases in which GX failed to yield Ct values were plotted with Ct values of 48. Red dots indicate cases with the G29179T mutation.

Figure 3

Phylogeny of Nextclade and Pangolin lineages. Phylogenetic tree of SARS-CoV-2 showing the distribution of sequences from 33 cases in this study. Each strain classified by Pangolin Lineage is the same color-coded as in Table 1. The numbers also represent the case numbers as listed in Table 1.